2 Microdialysis for Vitreal Pharmacokinetics |
|
|
23 |
||
EPITHELIAL BARRIER |
TISSUE BOUNDARY |
SOLID PHASE |
BLOOD VESSEL |
||
strong |
|
continuous |
low |
high |
fenestrated |
weak |
|
porous |
|
|
complete |
|
|
|
|
|
tight |
|
|
Diffusion resistance |
|||
MUSCLE TARGET SITE |
|
|
|
CIRCULATING FLUID |
|
|
|
|
|
||
|
|
|
|
|
FLUID FLOW |
|
|
|
|
|
ACTIVE TRANSPORT |
|
|
|
|
s |
|
d |
|
|
|
|
|
|
|
z |
|
|
|
|
i |
h |
|
|
|
|
|
|
|
|
|
c |
|
l |
|
|
v |
a |
|
|
|
||
|
|
|
|
|
|
Fig. 2.1 Diagrammatic representation of the routes of elimination of drugs from the vitreous of the eye
blood vessels. Movement of nutrient and waste molecules from the blood to retina is controlled by specific transport systems (Fig. 2.1). However, movement of these molecules such as amino acids and neurotransmitters to and within vitreous humor is via simple diffusion (Fig. 2.1) (Gunnarson et al. 1987). With respect to mass transfer, vitreous humor can be viewed as unstirred static fluid (Hughes et al. 1996). The globe is a closed system and does not allow sampling of tissues without irreversible damage. In such case, microdialysis plays an important role as a sampling technique which significantly reduces the number of animals required for pharmacokinetic studies.
2.3 Principle of Microdialysis
Microdialysis works on the principle of dialysis wherein a microdialysis probe is inserted in the tissue or fluid of interest. The probe has a semipermeable dialysis membrane which is circulated with physiological solution at a constant flow rate (Fig. 2.2).
24 |
R.D. Vaishya et al. |
Cos in ECF
Blood Capillary
Perfusate |
Dialysate |
Cic |
Ces |
|
Cec |
Fig. 2.2 Microdialysis schematics. Microenvironment within and surrounding the microdialysis probe in vivo. The solid and dashed line segments schematically represent the non-permeable probe wall and semipermeable membrane, respectively. Open and filled circles represent molecules of solute of interest and retrodialysis calibrator, respectively. Squares and triangles represent macromolecules which may bind solute and or calibrator but which are not recovered by dialysis. Arrows indicate the direction of transport
Following insertion of microdialysis probe, the concentration gradient across the semipermeable membrane causes the solute to diffuse in or out of dialysis probe. In order to avoid change in composition (ionic strength) of the surrounding fluid, the composition of perfusate should be similar to the fluid surrounding the dialysis membrane. Movement of solute molecules is also dependent on the molecular weight cut off (MWCO) of dialysis membrane. The process, governed by concentration gradient, never reaches the equilibrium since the perfusate is constantly circulated through the probe. Therefore, concentration in the dialysate is not same as that of vitreous. Analyte concentration in the dialyzing fluid (vitreous humor) can be determined from recovery, also known as extraction efficiency or relative recovery.
2.3.1 Extraction Efficiency/Recovery
Recovery is a ratio between the concentration of analyte in dialysate (Cout) and fluid surrounding the probe (Cin). The concentration of analyte in dialysate is a fraction of that present in the fluid surrounding the probe. Therefore, in vitro probe recovery is a key parameter for analyzing in vivo microdialysis data. In vitro probe recovery may be calculated by (2.1).
C
Recoveryin vitro = Cout . (2.1) in
2 Microdialysis for Vitreal Pharmacokinetics |
25 |
Following determination of recovery with defined parameters such as flow rate, (2.2) can be utilized to transform dialysate concentration ( Cˆout ) into actual vitreous concentration ( Cˆin ).
|
|
ˆ |
|
|
ˆ |
= |
Cout |
. |
(2.2) |
Cin |
Recoveryin vitro |
|
||
|
|
|
|
Absolute recovery is the amount of analyte over a definite period of time. It is the product of relative recovery (R), flow rate (F) and concentration of the analyte (C) (Wages et al. 1986).
Relative recovery can also be calculated by retrodialysis. In this method, an internal standard is perfused through dialysis tube and the loss in internal standard is measured along with the analyte from the dialysate. Recovery of both analyte and internal standard are calculated. Recovery, fractional loss of internal standard during dialysis, is calculated with (2.3).
Recovery |
|
= |
Cin -Cout |
, |
(2.3) |
internal standard |
|
||||
|
|
Cin |
|
||
|
|
|
|
||
Cin is the concentration of internal standard entering probe; Cout is the concentration of internal standard exiting the probe. Recovery of the internal standard and analyte can be compared taking ratio, given in (2.4).
Recoveryratio = |
Recoveryinternal standard |
, |
(2.4) |
|
|||
|
Recoveryanalyte |
|
|
A number of factors may influence in vitro probe recovery including perfusate flow rate and composition, temperature, properties of the membrane, probe design, analyte concentration and molecular weight. For example, the relative recovery decreases as the perfusate flow rate is raised (Wages et al. 1986). Among these, temperature and flow rate of perfusate are most critical factors influencing in vitro recovery. Wang et al. studied the relationship between perfusate flow rate and in vitro recovery utilizing zidovudine (AZT) as analyte and 3¢-azido-2¢,3¢- dideoxyuridine (AZdU) as internal standard. Recovery decreased exponentially with the increase in flow rate (Wang et al. 1993) (Fig. 2.3). Therefore, the rate of perfusate in dialysis probe is a key parameter that needs to be considered while optimizing microdialysis parameters. Usually a flow rate of 2 mL/min is preferred for most experiments. Figure 2.4 explains the influence of temperature on recovery at different perfusate flow rates (Wages et al. 1986). Recovery of DOPAC was studied by retrodialysis and effect of temperature on recovery was examined. Recovery was highest at 37°C and least at 23°C. This difference may be attributed to elevation in diffusion coefficient with rising temperature (Wages et al. 1986).
It has been well documented that in vivo recovery is always less than in vitro recovery during brain microdialysis studies (Amberg and Lindefors 1989). This may lead to misinterpretation of drug concentration data. During brain microdialysis, the
26 |
R.D. Vaishya et al. |
Fig. 2.3 Effect of flow rate on in vitro recovery of zidovudine (AZT) and loss of 3¢-azido-2¢,3¢- dideoxyuridine (AZdU) during microdialysis and retrodialysis. Filled square and circle represents loss of AZT and AZdU. Empty square and circle represents recovery of AZT and AZdU
100
|
90 |
|
(%) |
80 |
|
RECOVERY |
||
70 |
||
|
||
|
60 |
|
|
50 |
37°C
23°C
.04 .08 .12 .16 .20 .24 FLOW RATE ( L/MIN)
Fig. 2.4 Effect of temperature on in vitro recovery of DOPAC at different flow rates by retrodialysis
analyte concentration is measured in the extracellular fluid. Substrate diffuses from interstitial space in a tortuous path. Moreover, the analyte may partition inside the cells and therefore its concentration in the dialysate may not reflect the actual concentration when tissues are sampled with microdialysis. Movement through tortuous path and partitioning into cells may lower in vivo recovery of substrate.